Instrumental optimization of ruthenium(bpy)(3,3+)-based chemiluminescence and application as a detection strategy for capillary electrophoresis and micellar electrokinetic chromatography

Capillary electrophoresis (CE) has become a powerful separation tool recently. Among the detection methods available in CE, electrogenerated chemiluminescence (ECL) detection offers low detection limits with simple instrumentation. Electrogenerated Ru(bpy)₃3+-based CL has been used as a detection strategy for FIA, HPLC and CE to detect a variety of analytes including amines, amino acids, oxalate, various antihistamine drugs, and NADH.; In Chapter 2, Ru(bpy)₃3+-based CL detection in CE using a continuous in situ generation scheme is demonstrated and shown to provide advantages over other Ru3+ generation protocols. This detection strategy can be used to detect most amines and amino acids without derivatization, thus facilitating the optimization of the CE separation process independent of the detection method. The in situ cell described herein can operate continuously without the need to replenish the Ru(bpy)₃ 2+ reservoir and provides precise control of the electrochemical, light generation and detection processes. The important operational parameters for the in situ approach as applied to CE are identified and optimized, and the quantitative characteristics of this approach are evaluated.; In Chapter 3, the in situ generated Ru(bpy)₃3+-based CL detection cell is shown to be compatible with MEKC. The CL reagent, Ru(bpy) ₃2+ is continuously added postcapillary to avoid precipitating the anionic surfactant used to enhance the separation of neutral analytes. Ru(bpy)₃3+ is then electrochemically generated in situ at the interface between the separation capillary and the working electrode, where it can react with specific analytes, for example amines and amino acids to produce chemiluminescent emission. With this scheme, the critical micelle concentration is not exceeded in the detection zone, freeing the analyte to react with the Ru(bpy)₃3+ CL reagent. The separation and detection of various underivatized amines is demonstrated using this methodology. The experimental approaches used to improve the limit of detection while maintaining high separation efficiency are evaluated and discussed.; Two different designs for the Ru(bpy)₃3+-based CL detection cell were demonstrated and evaluated in Chapter 4. A bulk electrolysis generated Ru(bpy)₃3+ detection cell was designed and constructed where Ru(bpy)₃3+ is generated in the reagent capillary and delivered to the detection zone. The operational parameters and performance of the detection cell as compared to the in situ generated Ru(bpy)₃3+ detection cell, are optimized and discussed. The limitation of the in situ cell for the analysis of electrochemically active amines was investigated. An in situ generated Ru(bpy)₃ 3+ cell with a joint-less separation capillary was designed and constructed to eliminate this time consuming process and to improve cell-to-cell reproducibility. The performance of this new detection cell was demonstrated and its challenges and advantages are also discussed.; Application of the in situ generated Ru(bpy)₃3+ detection cell for the analysis of PTH and MTH amino acids is discussed in Chapter 5. The possibility of separating and detecting PTH and MTH amino acids by MEKC with Ru(bpy)₃3+-based CL detection was investigated. The limitation of this detection strategy for the analysis of PTH and MTH amino acids is addressed. Additional study of the stability of PTH and MTH amino acids, by CE with Ru(bpy)₃3+-based CL detection was performed at pH 9 and 10.